METABOLIC ENGINEERING RESEARCH OF HYALURONIC ACID-PRODUCING BACTERIUM Streptococcus Equi

A strain of Streptococcus equi which can produce hyaluronate capsule has been subjected to metabolic engineering research. And the pathway for its synthesis of hyaluronic acid (HA) was systematically studied.Firstly, the hyaluronate synthase (HAS) gene (hasA) was cloned into the cloning vector pUC19 by PCR using the total DNA sample of S.equi as template. And sequencing result showed its identity to that of has A from S.equisimilis recorded in Genebank. The expression vector pSE380-HAS was constructed and transformed into E.coli DH5a, expressing hasA gene by adding IPTG into the broth. And HA molecules were synthesized in vitro utilizing excessive active UDP-GLcA and UDP-GlcNAc sugar nucleotide precursors in the presence of a divalent Mg2+or Mn2+ ion.Secondly, the hyaluronate lyase gene (hyl) was cloned into the vector pUC19 by PCR using the total ONA sample ofS.equi as template and partially sequenced, too. The expression vector pSE380-/iy/ was constructed and transformed into E.coli DH5a, expressing hyl gene by adding IPTG into the broth .The expression of hyl gene showed a 120KDa protein band on SDS-PAGE gel and was found to have capability to degrade HA molecules derived from a microorganism dissolved in 0.1 M acetate buffer solution (pH4.0).Thirdly, the hyl gene was cloned to pUC19 and pBR322 respectively. Then the fragment containing Ampr derived from pUC19 and hasA came from pSE380-HAS were inserted into hyl either or both in vitro. Transformed the recombinated vectors into S.equi by electro-operation, then plating on HAG solid medium containing ampicillin , and selected clones hi which the hyl gene was inactivated by gene replacement through homologous recombination.Finally, we get some transformants with lower Hyl activity.